The present invention relates to plant biotechnology and specifically to a novel transformation protocol for obtaining transgenic turnip rape plants.
Turnip rape (Brassica rapa ssp. oleifera, syn. B. campestris) is general oil producer crop in the Northern Europe, Canada and Indian subcontinent. Canola type turnip rape oil has a high nutritional quality. Seeds of turnip rape are rich in storage proteins, which enables there usage as forage. Due to high industrial importance, turnip rape is often involved in transgenic investigations. However, there are not many reports about successful genetic transformation of the plant.
Transformation protocols using Agrobacterium infection have been developed for hypocotyl explants of turnip rape, B. rapa ssp. oleifera (Radke et al. 1992) and brown sarson, B. campestris (Mukhopadhyay et al. 1992). Some other reports on Agrobacterium mediated transformation of turnip rape (Knutzon et al. 1992; Shiba et al. 1995) are based on the protocol of Radke et al. 1992 (supra). Shoot regeneration on turnip rape explants has been very problematic and was the main obstacle which delayed development of transformation protocols in the 1980""s. Recently, recalcitrancy of B. rapa ssp. oleifera was overcome by using silver nitrate in the cultural medium during shoot regeneration of hypocotyl and cotyledon explants (Chi et al. 1990; Hachey et al. 1991; Palmer 1992).
Transformation protocols for explants of mature plants of a close relative of turnip rape, namely oilseed rape (Brassica napus), have been successfully developed (Fry et al. 1987; Pua et al. 1987; Radke et al. 1987). U.S. Pat. No. 5,188,958 for Moloney and Radke claims transformation of Brassica species. In the specification successful transformation has been shown for Brassica napus (cv. Westar) only.
Our specific aim is to use mature plant tissue, especially inflorescence carrying stem segments as an explant source enabling us to successfully propagate and transform the same plant genotype. In preliminary experiments we have examined different factors, which could have an effect on regeneration and transformation capacities of different explants of a mature plant. As a result of the experiments we have developed a new transformation protocol for inflorescence carrying stem segments of mature turnip rape plants using Agrobacterium tumefaciens infection.
The present invention thus provides a new and efficient transformation protocol of mature plants of turnip rape, in which protocol an internode section of the inflorescence carrying stem of a mature turnip rape plant is excised, the section is sterilized and cut into segments, which are then placed in a horizontal position on an agar cultivation medium (MS-medium) which is supplemented with silver nitrate and a hormone. Sucrose is used in all MS culturing media. The segments are cultivated on said medium for 1 day (24 hours) and then immersed in a MS solution inoculated with Agrobacterium tumefaciens bacteria carrying at least one gene foreign to said turnip rape. The extra liquid on the immersed segments is removed with a filter paper and the explants are placed in a horizontal position on a MS agar co-cultivation medium supplemented with hormones and optionally with acetosyringone, and co-cultivated with Agrobacteria for 2 days (48 hours) so as the transformation to be effected. The co-cultivated segments are then washed from the Agrobacteria and placed immediately in a vertical position with the basal side down on MS agar for selection with antibiotics. Selection is carried out for 3 to 6 weeks using kanamycin or hygromycin. Subsequently the stem segments are placed on a regeneration medium supplemented with hormones, and the transgenic shoots regenerated are separated, and grown for about 1 month on a MS medium without hormones. The main steps of the transformation protocol of the invention are given in FIG. 1.